Prior to the present invention, proteins such as antibodies have been radiolabeled with iodine. A more attractive approach to the radiolabeling of amines, polypeptide chains and proteins has been the "bifunctional chelate" methods in which a strong chelating group is covalently bonded to the protein which, in turn, is labeled with a variety of chelatable radionuclides. In the resultant product, the protein retains its biological function and the product also retains the radionuclide. When the chelating group chosen forms stable chelates, the radiolabel is likely to be stable in vivo. In addition, its presence will have only a minor effect on the specificity or other functional biochemical characteristics of the protein.
Attempts have been made to develop useful bifunctional chelates. Sundberg et al [J. Med. Chem., 17:1304 (1974)] was concerned with synthesizing 1-(p-benzyldiazonium)-EDTA as a chelating agent. Their method ultimately provided a labeled protein but the 7-step synthesis of the EDTA (ethylenediaminetetraacetic acid) intermediate makes this approach very unattractive. Yeh et al [J. Radioanalytical Chem., 53:327 (1979)] developed a method using 1-(p-carboxymethoxybenzyl)-EDTA as the linking molecule but without avoiding similar disadvantages. Others [Krejcarek et al, Biochem. Biophys. Res. Comm., 77:581 (1977); and Wagner et al, J. Nucl. Med., 20:428 (1979)] developed a method of linking diethylenetriaminepentaacetic acid (DTPA) to proteins via the synthesis of a mixed carboxycarbonic anhydride intermediate. The synthesis of this intermediate, although less complicated than others, is nevertheless not simple; the coupling reaction alone requires about 12 hours duration and must be followed by several purification steps requiring several days' time. Gluteraldehyde has also been considered as a method of coupling chelating agents to proteins [Pritchard et al, Proc. C. Soc. Exp. Biol. Med., 151:297 (176); Yokoyama et al, J. Nucl. Med., 22:32 (1981)], but the formation of dimers and the need to reduce the product in order to improve its stability in vivo are major disadvantages.
Overall, therefore, there is a long demonstrated need for a simple and efficient bifunctional chelating agent for amines, polypeptides and proteins and other medically important compounds such that their radiolabeled forms are stable in vivo and retain their specific biological activities to a substantial degree.